Our proposed work is aimed at understanding the expression of proinsulin. Previously we have identified the mRNA encoding fish proinsulin and this mRNA has been partially purified. We are now engaged in preparing a cDNA from this mRNA in order to use it as a probe for studying the expression of proinsulin mRNA biosynthesis under a variety of physiological conditions. Attempts will be made to prepare a double stranded DNA molecule for studies involving the structure of the gene itself. We are also currently studying the detailed mechanisms by which the preforms of secretory proteins are processed and extracellularly released. We will try to dissect out the steps involved in the preprotein reaction with a variety of protease inhibitors. Ultimately these inhibitors will be incubated with pancreatic islets in efforts to relate the secretion of proinsulin with cleavage of its preportion. Also, we are attempting to prepare a reconstituted model membrane system containing preprotein cleavage activity. Thus, from such studies, we will acquire new information concerning the biology of proinsulin expression in islet tissue, i.e., the molecular mechanisms by which a select class of effector molecules stimulate insulin synthesis; and the possible involvement of such effects in disease.